HIV is a complex retrovirus containing a number of genes not commonly found in other retroviruses. We are focusing on the analysis of two of these genes, vpu and vif. Vpu is encoded only by HIV-1 and encodes for a small integral membrane protein. Vpu is phosphorylated by casein kinase II at two highly conserved serine residues. Vpu regulates two biological functions: (i) enhancement of particle release, and (ii) degradation of CD4. To study the function of Vpu, we constructed a series of mutants and analyzed their effect both with regard to Vpu- mediated enhancement of particle release as well as CD4 degradation. We found that phosphorylation of Vpu was absolutely essential for CD4 degradation while unphosphorylated Vpu still retained at least 50% of wild type activity with regard to particle release. Phosphorylation of Vpu did not affect stability or intracellular distribution of Vpu. Thus, the two biological functions of Vpu are differentially regulated by phosphorylation. To study the importance of the Vpu TM domain for its function, we created a mutant containing a scrambled TM domain, VpuRD. In contrast to the Vpu phosphorylation mutant, VpuRD was still capable of inducing CD4 degradation although at a slightly reduced rate. However, this mutant was no longer able to support virus release from the cells. Preliminary evidence suggests that the Vpu TM domain forms an ion pore. It is thus possible, that virus release is regulated by an ion channel function of Vpu. In contrast, additional studies investigating the mechanism of CD4 degradation demonstrate that this process involves a direct interaction between Vpu and CD4. In summary, our data suggest that Vpu has two biological functions that are executed in different cellular compartments, rely on different functional domains of Vpu, and are mechanistically unrelated. Vif is a 23 kDa basic protein which has an important function in regulating infectivity of progeny virions. The biochemical mechanism of Vif function is obscure. We analyzed the role of Vif by studying its subcellular distribution by cell fractionation as well as confocal microscopy. We found that approximately half of intracellular Vif protein is associated with the cytoskeleton, specifically intermediate filaments. The association of Vif with intermediate filaments is specific and can result in the reorganization of the cytoskeletal network. Experiments are ongoing to study the significance of this finding and to correlate it with the observed effects of Vif on viral infectivity.